The present invention relates to arylsulfonyl hydroxamic acid derivatives which are inhibitors of matrix metalloproteinases or the production of tumor necrosis factor (hereinafter also referred to as TNF) and as such are useful in the treatment of a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis bullosa, sceritis and other diseases characterized by matrix metalloproteinase activity, as well as AIDS, sepsis, septic shock and other diseases involving the production of TNF.
This invention also relates to a method of using such compounds in the treatment of the above diseases in mammals, especially humans, and to the pharmaceutical compositions useful therefor.
There are a number of enzymes which effect the breakdown of structural proteins and which are structurally related metalloproteases. Matrix-degrading metalloproteinases, such as gelatinase, stromelysin and collagenase, are involved in tissue matrix degradation (e.g. collagen collapse) and have been implicated in many pathological conditions involving abnormal connective tissue and basement membrane matrix metabolism, such as arthritis (e.g. osteoarthritis and rheumatoid arthritis), tissue ulceration (e.g. corneal, epidermal and gastric ulceration), abnormal wound healing, periodontal disease, bone disease (e.g. Paget""s disease and osteoporosis), tumor metastasis or invasion, as well as HIV-infection (J. Leuk. Biol., 52 (2): 244-248, 1992).
Tumor necrosis factor is recognized to be involved in many infectious and auto-immune diseases (W. Friers, FEBS Letters, 1991, 285, 199). Furthermore, it has been shown that TNF is the prime mediator of the inflammatory response seen in sepsis and septic shock (C. E. Spooner et al., Clinical Immunology and Immunopathology, 1992, 62 S11).
The present invention relates to a compound of the formula 
or the pharmaceutically acceptable salt thereof, wherein the broken line represents an optional double bond;
Y is carbon, oxygen, sulfur, sulfoxide, sulfone or nitrogen;
R1, R2, R3, R4, R5, R6, R7, R8 and R9 are selected from the group consisting of hydrogen, (C1-C6)alkyl optionally substituted by (C1-C6)alkylamino, (C1-C6,)alkylthio, (C1-C6)alkoxy, trifluoromethyl, (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)arylamino, (C6-C10)arylthio, (C6-C10)aryloxy, (C5-C9)heteroarylamino, (C5-C9)heteroarytti, (C5-C9)heteroaryloxy, (C6-C10)aryl(C6-C10)aryl, (C3-C6)cycloalykyl, hydroxy(C1-C6)alkyl, (C1-C6)alkyl(hydroxymethylene),piperazinyl,(C6-C10)aryl(C1-C6)alkoxy,(C5-C9)heteroaryl(C1-C6)alkoxy, (C1-C6)acylamino, (C1-C6)acylthio, (C1-C6)acyloxy, (C1-C6)alkylsulfinyl, (C6-C10)arylsulfinyl, (C1-C6)alkylsulfonyl, (C6-C10)arylsulfonyl, amino, (C1-C6)alkylamino or ((C1-C6)alkylamino)2; (C2-C6)alkenyl, (C6-C10)aryl(C2-C6)alkenyl, (C5-C9)heteroaryl(C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl(C2-C6)alkynyl, (C5-C9)heteroaryl(C2-C6)alkynyl, (C1-C6)alkylamino, (C1-C6)alkylthio, (C1-C6)alkoxy, trifluoromethyl, (C1-C6)alkyl(difluoromethylene), (C1-C3)alkyl(difluoromethylene)(C1-C3)alkyl, (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)arylamino, (C6-C10)arylthio, (C6-C10)aryloxy, (C5-C9)heteroarylamino, (C5-C9)heteroarytthio, (C5-C9)heteroaryloxy, (C3-C6)cycloalkyl, (C1-C6)alkyl(hydroxymethylene), piperidyl, (C1-C6)alkylpiperidyl, (C1-C6)acylamino, (C1-C6)acylthio, (C1-C6)acyloxy, R13(C1-C6)alkyl wherein R13 is (C1-C6)acylpiperazino, (C6-C10)arylpiperazino, (C5-C9)heteroarylpiperazino, (C1-C6)alkylpiperazino, (C1-C10)aryl(C1-C6)alkylpiperazino,(C5-C9)heteroaryl(C1-C6)alkylpiperazino,morpholino,thiomorpholino, piperidino, pyrrolidino, piperidyl, (C1-C6)alkylpiperidyl, (C6-C10)arylpiperidyl, (C5-C9)heteroarylpiperidyl, (C1-C6)alkylpiperidyl(C1-C6)alkyl, (C6-C10)arylpiperidyl(C1-C6)alkyl, (C5-C9)heteroarylpiperidyl(C1-C6)alkyl or (C1-C6)acylpiperidyl;
or a group of the formula 
xe2x80x83wherein
n is 0 to 6;
Z is hydroxy, (C1-C6)alkoxy or NR14R15 wherein R14 and R15 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl optionally substituted by (C1-C6)alkylpiperidyl, (C6-C10)arylpiperidyl, (C5-C9)heteroarylpiperidyl, (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)aryl(C6-C10)aryl or (C3-C6)cycloalkyl; piperidyl, (C1-C6)alkylpiperidyl, (C6-C10)arylpiperidyl, (C5-C9)heteroarylpiperidyl, (C1-C6)acylpiperidyl, (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)aryl(C6-C10)aryl(C3-C6)cycloalkyl, R16(C2-C6)alkyl, (C1-C5)alkyl(CHR16)(C1-C6)alkyl wherein R16 is hydroxy, (C1-C6)acyloxy, (C1-C,)alkoxy, piperazino, (C1-C6)acylamino, (C1-C6)alkylthio, (C6-C10)arylthio, (C1-C6)alkylsulfinyl, (C6-C10)arylsulfinyl, (C1-C6)alkylsulfoxyl, (C6-C10)arylsulfoxyl, amino, (C1-C6)alkylamino, ((C1-C6)alkyl)2amino, (C1-C6)acylpiperazino, (C1-C6)alkylpiperazino, (C6-C10)aryl(C1-C6)alkylpiperazino, (C5-C9)heteroaryl(C1-C6)alkylpiperazino,morpholino,thiomorpholino, piperidino or pyrrolidino; R17(C1-C6)alkyl, (C1-C5)alkyl(CHR17)(C1-C6)alkyl wherein R17 is piperidyl or (C1-C6)alkylpiperidyl; and CH(R18)COR19 wherein R18 is hydrogen, (C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl, (C1-C6)alkylthio(C1-C6)alkyl, (C6-C10)arylthio(C1-C6)alkyl, (C1-C6)alkylsulfinyl(C1-C6)alkyl, (C6-C10)arylsulfinyl(C1-C6)alkyl, (C1-C6)alkylsulfonyl(C1-C6)alkyl, (C6-C10)arylsulfonyl(C1-C6)alkyl, hydroxy(C1-C6)alkyl, amino(C1-C6)alkyl, (C1-C6)alkylamino(C1-C6)alkyl, ((C1-C6)alkylamino)2(C1-C6)alkyl, R20R21NCO(C1-C6)alkyl or R20OCO(C1-C6)alkyl wherein R20 and R21 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkyl and (C5-C9)heteroaryl(C1-C6)alkyl; and R19 is R22O or R22R23N wherein R22 and R23 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkyl and (C5-C9)heteroaryl(C1-C6)alkyl;
or R14 and R15, or R20 and R21, or R22 and R23 may be taken together to form an azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, indolinyl, isoindolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, (C1-C6)acylpiperazinyl, (C1-C6)alkylpiperazinyl, (C6-C10)arylpiperazinyl, (C5-C9)heteroarylpiperazinyl or a bridged diazabicydoalkyl ring selected from the group consisting of 
xe2x80x83wherein
r is 1, 2 or 3;
m is 1 or 2;
p is 0 or 1; and
Q is hydrogen, (C1-C3)alkyl, (C1-C6)acyl or (C1-C6)alkoxy carbamoyl;
or R1 and R2, or R3 and R4, or R5 and R6 may be taken together to form a carbonyl;
or R1 and R2, or R3 and R4, or R5 and R6, or R7 and R8 may be taken together to form a (C3-C6)cycloalkyl, oxacyclohexyl, thiocyclohexyl, indanyl or tetralinyl ring or a group of the formula 
xe2x80x83wherein
R24 is hydrogen, (C1-C6)acyl, (C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl or (C1-C6)alkylsulfonyl; and
Ar is (C6-C10)aryl or (C5-C9)heteroaryl, each of which may be optionally substituted by (C1-C6)alkyl, one or two (C1-C6)alkoxy, (C6-C10)aryloxy or (C5-C9)heteroaryloxy;
with the proviso that R7 is other than hydrogen only when R8 is other than hydrogen;
with the proviso that R6 is other than hydrogen only when R5 is other than hydrogen;
with the proviso that R3 is other than hydrogen only when R4 is other than hydrogen;
with the proviso that R2 is other than hydrogen only when R1 is other than hydrogen;
with the provisio that when R1, R2 and R9 are a substituent comprising a heteroatom, the heteroatom cannot be directly bonded to the 2- or 6-positions;
with the proviso that when X is nitrogen, R4 is not present;
with the proviso that when X is oxygen, sulfur, sulfoxide, sulfone or nitrogen and when one or more of the group consisting of R1, R2, R5 and R6, is a substituent comprising a heteroatom, the heteroatom cannot be directly bonded to the 4- or 6-positions;
with the proviso that when Y is oxygen, sulfur, sulfoxide, sufone or nitrogen and when one or more of the group consisting of R3, R4, R7 and R8, are independently a substituent comprising a heteroatom, the heteroatom cannot be directly bonded to the 3- or 5-positions;
with the proviso that when X is oxygen, sulfur, sulfoxide or sulfone, R3 and R4 are not present;
with the proviso that when Y is nitrogen, R4 is not present;
with the proviso that when Y is oxygen, sulfur, sulfoxide or sufone, R5 and R6 are not present;
with the proviso that when Y is nitrogen, R6 is not present;
with the proviso that when the broken line represents a double bond, R4 and R6 are not present;
with the proviso that when R3 and R5 are independently a substituent comprising a heteroatom when the broken line represents a double bond, the heteroatom cannot be directly bonded to positions X and Y;
with the proviso that when either the X or Y position is oxygen, sulfur, sulfoxide, sulfone or nitrogen, the other of X or Y is carbon;
with the proviso that when X or Y is defined by a heteroatom, the broken line does not represent a double bond;
with the proviso that when R1, R2, R3, R4, R5, R6, R7, R8 and R9 are all defined by hydrogen or (C1-C6)alkyl, either X or Y is oxygen, sulfur, sulfoxide, sulfone or nitrogen, or the broken line represents a double bond.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties or combinations thereof.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, includes O-alkyl groups wherein xe2x80x9calkylxe2x80x9d is defined above.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluoro, chloro, cyano, nitro, trifluoromethyl, (C1-C6)alkoxy, (C6-C10)aryloxy, trifluoromethoxy, difluoromethoxy and (C1-C6)alkyl.
The term xe2x80x9cheteroarylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic heterocyclic compound by removal of one hydrogen, such as pyridyl, furyl, pyroyl, thienyl, isothiazolyl, imidazolyl, benzimidazolyl, tetrazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, isobenzofuryl, benzothienyl, pyrazolyl, indolyl, isoindolyl, purinyl, carbazolyl, isoxazolyl, thiazolyl, oxazolyl, benzthiazolyl or benzoxazolyl, optionaly substituted by 1 to 2 substituents independently selected from the group consisting of fluoro, chloro, trifluorometyl. (C1-C6)alkoxy, (C6-C10)aryloxy, trifluoromethoxy, difluoromethoxy and (C1-C6)alkyl.
The term xe2x80x9cacylxe2x80x9d, as used herein, unless otherwise indicated, includes a radical of the general formula RCO wherein R is alkyl, alkoxy, aryl, arylalkyl or arylalkyloxy and the terms xe2x80x9calkylxe2x80x9d or xe2x80x9carylxe2x80x9d we as defined above.
The term xe2x80x9cacyloxyxe2x80x9d, as used herein, includes O-acyl groups wherein xe2x80x9cacylxe2x80x9d is defined above.
The positions on the ring of formula 1, as used herein, are defined as follows: 
The preferred conformation of the compound of formula I includes hydroxamic acid axially disposed in the 2-position.
The compound of formula I may have chiral centers and therefore exist in different enantiomeric forms. This invention relates to all optical isomers and stereoisomers of the compounds of formula I and mixtures thereof.
Preferred compounds of formula I include those wherein Y is oxygen, nitrogen or sulfur.
Other preferred compounds of formula I include those wherein Ar is 4-methoxyphenyl or 4-phenoxyphenyl.
Other preferred compounds of formula I include those wherein R8 is (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)aryl(C1-C6)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl, carboxylic acid carboxylic acid (C1-C6)alkyl.
Other preferred compounds of formula I include those wherein R2, R3, R6, R7 and R9 are hydrogen.
More preferred compounds of formula I include those wherein Y is carbon, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R8 is (C6-C10)arylalkenyl or (C5-C9)heteroaryalkynyl.
More preferred compounds of formula I include those wherein Y is oxygen, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R8 is (C6-C10)arylalkenyl or (C5-C9)heteroarykynyl.
More preferred compounds of formula I include those wherein Y is carbon, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R8 is carboxylic acid or carboxylic acid (C1-C6)alkyl.
More preferred compounds of formula I include those wherein Y is oxygen, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R8 is carboxylic acid or carboxylic acid (C1-C6)alkyl.
More preferred compounds of formula I include those wherein Y is carbon, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R5 is (C6-C10)arylalkenyl or (C5-C9)heteroarylalkynyl.
More preferred compounds of formula I include those wherein Y is oxygen, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R5 is (C6-C10)arylalkenyl or (C5-C9)heteroarylalkynyl.
More preferred compounds of formula I include those wherein Y is carbon, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R5 is carboxylic acid or carboxylic acid (C1-C6)alkyl.
More preferred compounds of formula I include those wherein Y is oxygen, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R5 is carboxylic acid or carboxylic acid (C1-C6)alkyl.
More preferred compounds of formula I include those wherein Y is carbon, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R5 is (C1-C6)alkylamino.
More preferred compounds of formula I include those wherein Y is oxygen, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R8 is (C1-C6)alkylamino.
Specific preferred compounds of formula I include the following:
(2R,3S)-N-hydroxy-3ethynyl-1-(4-methoxybenzenesulfonyl)-piperidine-2-carboxamide;
(2R,3S)-N-hydroxy-1-(4-methoxybenzenesulfonyl)-3-(5-methoxythiophene-2yl-ethynyl)-piperidine-2 carboxamide;
(2R,3R)-N-hydroxy-1-(4-methoxybenzenesulfonyl)-3-(3-pyridin-3-yl-prop-2-ynyl)-piperidine2-carboxamide;
(2S,3R)-N-hydroxy-4-(4-methoxybenzonesulfonyl)-2-pyridine-3-yl-morpholine-3-carboxamide;
(2S,3R)-N-hydroxy-2-hydroxycarbamoyl-4-(4-methoxybenzenesulfonyl)-morpholine-3-carboxamide;
(2R,3R)-N-hydroxy-2-hydroxycarbamoyl-4-(4-methoxybenzenesulfonyl)-piperidine-2-carboxamide;
(2R,3S)-N-hydroxy-1-(4-methoxybenzenesulfonyl)-3-(4-phenylpyridine-2-yl)-piperidine-2-carboxamide;
(2S,3R)-N-hydroxy-1-(4-methoxybenzenesulfonyl)-2-(4-phenylpyridine-2-yl)-morpholine-2-carboxamide;
(2R,3S)-N-hydroxy-3-(2-chloro-4-fluorophenyl)-1-(4-methoxybenzenesulfonyl)-piperidine-2-carboxamide; and
(2S,3R)-N-hydroxy-2-(2-chloro-4-fluorophenyl)-1-(4-methoxybenzenesulfonyl)-piperidine-3-carboxamide.
The present invention also relates to a pharmaceutical composition for (a) the treatment of a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis bullosa, scleritis and other diseases characterized by matrix metalloproteinase activity, AIDS, sepsis, septic shock and other diseases involving the production of tumor necrosis factor (TNF) or (b) the inhibition of matrix metalloproteinases or the production of tumor necrosis factor (TNF) in a mammal, including a human, comprising an amount of a compound of claim 1 or a pharmaceutically acceptable sat theeof, effective in such treatments or inhibition and a pharmaceutically acceptable carrier.
The present invention also relates to a method for the inhibition of (a) matrix metalloproteinases or (b) the production of tumor necrosis factor (TNF) in a mammal, including a human, comprising administering to said mammal an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.
The present invention also relates to a method for treating a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis bullosa, scleritis and other diseases characterized by matrix metalloproteinase activity, AIDS, sepsis, septic shock and other diseases involving the production of tumor necrosis factor (TNF) in a mammal, inducing a human, comprising administering to said mammal an amount of a compound of claim 1 or a pharmaceutically acceptable sat thereof, effective in treating such a condition.
The following reaction Schemes illustrate the preparation of the compounds of the present invention. Unless otherwise indicated R1, R2, R3, R4, R5, R6, R7, R8, R9, n and Ar in the reaction Schemes and the discussion that follow are defined as above. 
In reaction 1 of Preparation 1 the compound of formula XVI is converted to the corresponding hydroxy ester compound of formula VI by first reacting XVI with an arylsulfonylhalide in the presence of trithylamine and an aprotic solvent, such as methylene chloride, tetrahydrofuran or dioxane, at a temperature between about 20xc2x0 C. to about 30xc2x0 C., preferably at room temperature. The compound so formed is further reacted with a compound of the formula 
wherein R25 is carbobenzyloxy, (C1-C6)alkyl, benzyy, allyl or tert-butyl, in the presence of sodium hexamethyidisilazane and a tetrahydrofuran-dimethylformamide solvent mixture at a temperature between about xe2x88x9220xc2x0 C. to about 20xc2x0 C., preferably about 0xc2x0 C., to form the hydroxy ester compound of formula VI.
In reaction 1 of Preparation 2, the amine compound of formula XVIII, wherein R25 is as defined above, is converted to the corresponding arylsulfonyl amine compound of formula XVII by (1) reacting XVIII with an arylsulfonylhalide in the presence of triethylamine and an aprotic solvent, such as methylene chloride, tetrahydrofuran, or dioxane, at a temperature between about 20xc2x0 C. to about 30xc2x0 C., preferably at room temperature, (2) reacting the compound so formed with a compound of the formula 
in the presence of sodium hexamethyidisilazane and a tetrahydrofuran-dimethylformamide solvent mixture at a temperature between about xe2x88x9220xc2x0 C. to about 20xc2x0 C., preferably about 0xc2x0 C., and (3) further reacting the compound so formed with ozone in a methylene chloride-methanol solution at a temperature between about xe2x88x9290xc2x0 C. to about xe2x88x9270xc2x0 C., preferably about xe2x88x9278xc2x0 C. The unstable ozonide compound so formed is then reacted with triphenylphosphine to form the arylsulfonyl amine compound formula XVII. In Reaction 2 of Preparation 2, the arylsulfonyl amine compound of formula XVII is converted to the corresponding hydroxy ester compound of formula VI by reacting XVII with a compound of the formula 
wherein W is lithium, magnesium, copper or chromium.
In reaction 1 of Scheme 1. the compound of formula VI, wherein the R25 protecting group is carbobenzyloxy, (C1-C6) alkyl, benzyl, allyl or tert-butyl, is converted to the corresponding morpholinone compound of formula V by lactonization and subsequent Claisen rearrangement of the compound of formula VI. The reaction is facilitated by the removal of the R25 protecting group from the compound of formula VI is carried out under conditions appropriate for that particular R25 protecting group in use. Such conditions include: (a) treatment with hydrogen and a hydrogenation catalyst, such as 10% palladium on carbon, where R25 is carbobenzyloxy, (b) saponification where R25 is lower alkyl, (c) hydrogenolysis where R25 is benzyl, (d) treatment with a strong acid, such as trifluoroacetic acid or hydrochloric acid, where R25 is tert-butyl, or (e) treatment with tributyitinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride where R25 is allyl.
In reaction 2 of Scheme 1, the morpholinone compound of formula V is converted to the carboxylic acid compound of formula IV by reacting V with lithium hexamethyidisilazane in an aprotic solvent, such as tetrahydrofuran, at a temperature between about xe2x88x9290xc2x0 C. to about xe2x88x9270xc2x0 C., preferably about xe2x88x9278xc2x0 C. Trimethylsilyl chloride is then added to the reaction mixture and the solvent, tetrahydrofuran, is removed in vacuo and replaced with toluene. The resulting reaction mixture is heated to a temperature between about 100xc2x0 C. to about 120xc2x0 C., preferably about 110xc2x0 C., and treated with hydrochloric acid to form the carboxylic acid compound of formula IV.
In reaction 3 of Scheme 1, the carboxylic acid compound of formula IV is converted to the corresponding hydroxamic acid compound of formula III by treating IV with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenztriazole in a polar solvent, such as dimethylformamide, followed by the addition of hydroxylamine to the reaction mixture after a time period between about 15 minutes to about 1 hour, preferably about 30 minutes. The hydroxylamine is preferably generated in situ from a salt form, such as hydroxylamine hydrochloride, in the presence of a base, such as N-methylmorpholine. Alternatively, a protected derivative of hydroxylamine or its salt form, where the hydroxyl group is protected as a tert-butyl, benzy or allyl ether, may be used in the presence of (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorphosphate and a base, such as N-methylmorpholine. Removal of the hydroxylamine protecting group is carried out by hydrogenolysis for a benzyl protecting group or treatment with a strong acid, such as trifluoroacetic acid, for a tert-butyl protecting group. The allyl protecting group may be removed by treatment with tributyttinhydride and acetic acid in the presence of catalytic bis(tiphenylphosphine) palladium (II) chloride. N,O-bis(4-methoxybenzyl)hydroxylamine may also be used as the protected hydroxylamine derivative where deprotection is achieved using a mixture of methanesulfonic acid and trifluoroacetic acid.
In reaction 4 of Scheme 1, the hydroxamic acid compound of formula III is converted, if desired, to the corresponding piperidine compound of formula II by treating III with hydrogen and a hydrogenation catayst, such a 10% palladium on carbon.
In reaction 1 of Scheme 2, the arylsulfonylpiperazine compound of formula IX, wherein R26 is carbobenzyloxy, benzyl or carbotertbutyloxy, is converted to the compound of formula VIII by reacting IX with a protected derivative of hydroxytamine of the formula
R27ONH2.HCl
wherein R27 is tert-butyl, benzyl or allyl, in the presence of dicyclohexylcarbodiimide, dimethylaminopyridine and an aprotic solvent, such as methylene chloride. The R26 protecting group is chosen such that it may be selectively removed in the presence of an without loss of the R27 protecting group, therefore, R26 cannot be the same as R27. Removal of the R26 protecting group from the compound of formula IX is carried out under conditions appropriate for that particular R26 protecting group in use. Such conditions include; (a) treatment with a hydrogen and a hydrogenation catalyst, such as 10% palladium on carbon, where R26 is carbobenzyloxy, (b) hydrogenolysis where R26 is benzyl or (c) treatment with a strong acid, such as trifluoroacefic acid or hydrochloric acid where R26 is carbotertbutyloxy.
In reaction 2 of Scheme 2, the compound of formula VIII is converted to the corresponding hydroxamic acid compound of formula VII, wherein R5 is hydrogen or (C1-C6)alkyl, by reacting, if desired, VIII with an alkylhalide when R5 is (C1-C6)alkyl. Subsequent removal of the R27 hydroxylamine protecting group is carried out by hydrogenolysis for a benzyl protecting group or treatment with a strong acid, such as trifluoroacetic acid, for a tert-butyl protecting group. The allyl protecting group may be removed by treatment with tributyttinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride.
In reaction 1 of Scheme 3, the aryisuffonylamine compound of formula XII, wherein R25 is as defined above, is converted to the corresponding piperizine compound of formula XI by reacting XII with a carbodiimide and a base, such as triethylamine. The compound of formula XI is further reacted to give the hydroxamic acid compound of formula X according to the procedure described above in reaction 3 of Scheme 1.
In reaction 1 of Scheme 4, removal of the R28 protecting group and subsequent reductive amination of the compound of formula XXII, wherein Y is oxygen, sulfur or carbon, to give the corresponding imine compound of formula XXI is carried out under conditions appropriate for that particular R28 protecting group in use. Such conditions include those used above for removal of the R26 protecting group in reaction 1 of Scheme 2.
In reaction 2 of Scheme 4 the imine compound of formula XXI is converted to the corresponding piperidine compound of formula XX by reacting XXI with a nucleophile of the formula R2M wherein M is lithium, magnesium halide or cerium halide. The reaction is carried out in ether solvents, such as diethyl ether or tetrahydrofuran, at a temperature between about xe2x88x9278xc2x0 C. to about 0xc2x0 C., preferably about xe2x88x9270xc2x0 C.
In reaction 3 of Scheme 4, the sulfonation of the piperidine compound of formula XX to given the corresponding arylsullonylpiperidine compound of formula XIX is carried out by reacting XX with an arylsulfonylhalide in the presence of triethylamine and an aprotic solvent, such as metherone chloride, tetrahydrofuran or dioxane, at a temperature between about 20xc2x0 C. to about 30xc2x0 C., preferably at room temperature.
In reaction 4 of Scheme 4, the arylsuffonylpiperidine compound of formula XIX is converted to the hydroxamic acid compound of formula XIX according to the procedure described above in reaction 3 of Scheme 1.
In reaction 1 of Scheme 5, the compound of formula XXVI, wherein the R29 and R31 protecting groups are each independently selected from the group consisting of carbobenzyloxy, benzyl and carbotertbutyloxy and R30 is carbobenzyloxy, (C1-C6)alkyl, benzyl, allyl or tert-butyl, is converted to the corresponding imine compound of formula XXV by the removal of the R29 protecting group and subsequent reductive amination of the compound of formula XXVI. The R29 protecting group is chosen such that it may be selectively removed in the presence of and without loss of the R31 protecting group. Removal of the R29 protecting group from the compound of formula XXVI is carried out under conditions appropriate for that particular R29 protecting group in use which will not affect the R31 protecting group. Such conditions include; (a) treatment with hydrogen and a hydrogenation catalyst, such as 10% palladium on carbon, where R29 is carbobenzyloxy and R31 is tert-butyl, (b) saponification where R29 is (C1-C6)alkyl and R31 is tert-butyl, (c) hydrogenolysis where R29 is benzyl and R31 is (C1-C6) alkyl or tert-butyl, (d) treatment with a strong acid such as trifluoroacetie acid or hydrochloric acid where R29 is tert-butyl and R31 is (C1-C6)alkyl, benzyl or allyl, or (e) treatment with tributyttinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride where R29 is allyl and R31 is (C1-C6)alkyl, benzyl or tert-butyl. The R30 protective group may be selected such that it is removed in the same reaction step as the R29 protecting group.
In reaction 2 of Scheme 5, the imine compound of formula XXV is converted to the corresponding compound of formula XXIV by reacting XXV with a nucleophile of the formula R2M wherein M is lithium, magnesium halide or calcium halide. The reaction is carried out in ether solvents, such as diethyl ether or tetrahydrofuran, at a temperature between about xe2x88x9278xc2x0 C. to about 0xc2x0 C., preferably about xe2x88x9270xc2x0 C.
In reaction 3 of Scheme 5, the sulfonation of the piperidine compound of formula XXIV to give the corresponding arylsulfonylpiperidine compound of formula III is carried out according to the procedure described above in reaction 3 of Scheme 4.
In reaction 4 of Scheme 5, the arylsulfonylpiperidine compound of formula XXIII is converted to the hydroxamic acid compound of formula XIV by (1) removing the R30, if needed, and R31 protecting groups from XXIII followed by (2) reacting XXIII according to the procedure described above in reaction 3 of Scheme 1. Removal of the R30 and R31 protecting groups from the compound of formula XXIII is carried out under conditions appropriate for that particular R30 and R31 protecting group in use. Such conditions include those used above for removal of the R25 protecting group in reaction 1 of Scheme 1.
Pharmaceutically acceptable salts of the acidic compounds of the invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium slats, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)methylammonium slats.
Similarly acid addition sats, such as of mineral acids, organic carboxylic and organic sulfonic acids e.g. hydrochloric acid, methanesuifonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure.
The ability of the compounds of formula I or their pharmaceutically acceptable salts (hereinafter also referred to as the compounds of the present invention) to inhibit matrix metalloproteinases or the production of tumor necrosis factor (TNF) and, consequently, demonstrate their effectiveness for treating diseases characterized by matrix metalloproteinase or the production of tumor necrosis factor is shown by the following in vitro assay tests.
Human recombinant collagenase is activated with trypsin using the following ratio: 10 xcexcg trypsin per 100 xcexcg of collagenase. The trypsin and collagenase are incubated at room temperature for 10 minutes then a five fold excess (50 xcexcg/10 xcexcg trypsin) of soybean trypsin inhibitor is added.
10 mM stock solutions of inhibitors are made up in dimethyl sulfoxide and then diluted using the following Scheme:
10 mMxe2x86x92120 xcexcMxe2x86x9212 xcexcMxe2x86x921.2 xcexcMxe2x86x920.12 xcexcM
Twenty-five microliters of each concentration is then added in triplicate to appropriate wells of a 96 well microfluor plate. The final concentration of inhibitor will be a 1:4 dilution after addition of enzyme and substrate. Positive controls (enzyme, no inhibitor) are set up in wells D1-D6 and blanks (no enzyme, no inhibitors) are set in wells D7-D12.
Collenase is diluted to 400 ng/ml and 25 xcexcl is then added to appropriate wells of the microfluor plate. Final concentration of collagenase in the assay is 100 ng/ml.
Substrate (DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2) is made as a 5 mM stock in dimethyl sulfoxide and then diluted to 20 xcexcM in assay buffer. The assay is initiated by the addition of 50 xcexcl substrate per well of the microfluor plate to give a final concentration of 10 xcexcM.
Fluorescence readings (360 nM excitation, 460 nm emission) were taken at time 0 and then at 20 minute intervals. The assay is conducted at room temperature with a typical assay time of 3 hours.
Fluorescence vs time is then plotted for both the blank and collagenase containing samples (data from triplicate determinations is averaged). A time point that provides a good signal (the blank) and that is on a linear part of the curve (usually around 120 minutes) is chosen to determine IC50 values. The zero time is used as a blank for each compound at each concentration and these values are subtracted from the 120 minute data. Data Is plotted as inhibitor concentration vs % control (inhibitor fluorescence divided by fluorescence of collagenase alonexc3x97100). IC50""s are determined from the concentration of inhibitor that gives a signal that is 50% of the control.
If IC50""s are reported to be  less than 0.03 xcexcM then the inhibitors are assayed at concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.03 xcexcM and 0.003 xcexcM.
Inhibition of gelatinase activity is assayed using the Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2 substrate (10 xcexcM) under the same conditions as inhibition of human collagenase (MMP-1).
72 kD gelatinase is activated with 1 mM APMA (p-aminophenyl mercuric acetate) for 15 hours at 4xc2x0 C. and is diluted to give a final concentration in the assay of 100 mg/ml. Inhibitors are diluted as for inhibition of human collagenase (MMP-1) to give final concentrations in the assay of 30 xcexcM, 3 xcexcM, 0.3 xcexcM and 0.03 xcexcM. Each concentration is done in triplicate.
Fluorescence readings (360 nm exciation, 460 emission) are taken at time zero and then at 20 minutes intervals for 4 hours.
IC50""s are determined as per inhibition of human collagenase (MMP-1). If IC50""s are reported to be less than 0.03 xcexcM, then the inhibitors we assayed at final concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.003 xcexcM and 0.003 xcexcM.
Inhibition of stromelysin acti is based on a modified spectrophotometric assay described by Weingarten and Feder (Weingarten, H. and Feder, J., Spectrophotometric Assay for Vertebrate Collagenase, Anal. Biochem. 147, 437-440 (1985)). Hydrolysis of the thio peptolide substrate [Ac-Pro-Leu-Gly-SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2H5] yields a mercaptan fragment that can be monitored in the presence of Eliman""s reagent.
Human recombinant prostromelysin is activated with trypsin using a ratio of 1 xcexcl of a 10 mg/ml bypsin stock per 26 xcexcg of stromelysin. The trypsin and stromelysin are incubated at 37xc2x0 C. for 15 minutes followed by 10 xcexcl of 10 mg/ml soybean trypsin inhibitor for 10 minutes at 37xc2x0 C. for 10 minutes at 37xc2x0 C. to quench trypsin activity.
Assays are conducted in a totW volume of 250 xcexcl of assay buffer (200 mM sodium chloride, 50 mM MES, and 10 mM calcium chloride, pH 6.0) in 96-well microliter plates. Activated stromelysin is diluted in assay buffer to 25 xcexcg/ml. Ellman""s reagent (3-Carboxy-4-nitrophenyl disulide) is made as a 1M stock in dimethyl formamide and diluted to 5 mM in assay buffer with 50 xcexcl per well yielding at 1 mM final concentration.
10 mM stock solutions of inhibitors are made in dimethyl sulfoxide and diluted serially in assay buffer such that addition of 50 xcexcL to the appropriate wells yields final concentrations of 3 xcexcM, 0.3 xcexcM, 0.003 xcexcM, and 0.0003 xcexcM. All conditions are completed in triplicate.
A 300 mM dimethyl sulfoxide stock solution of the peptide substrate is diluted to 15 mM in assay buffer and the assay is irntiated by addition of 50 xcexcl to each well to give a final concentration of 3 mM substrate. Blanks consist of the peptide substrate and Ellman""s reagent without the enzyme. Product formation was monitored at 405 nm with a Molecular Devices UVmax plate reader.
IC50 values were determined in the same manner as for collagenase.
Human recombinant MMP-13 is activated with 2 mM APMA (p-aminophenyl mercuric acetate) for 1.5 hours, at 37xc2x0 C. and is diluted to 400 mg/ml in assay buffer (50 mM Tris, pH 7.5, 200 mM sodium chloride, 5 mM calcium chloride, 20 xcexcM zinc chloride, 0.02% brij). Twenty-five microliters of diluted enzyme is added per well of a 96 well microfluor plate. The enzyme is then diluted in a 1:4 ratio in the assay by the addition of inhibitor and substrate to give a final concentration in the assay of 100 mg/ml.
10 mM stock solutions of inhibitors are made up in dimethyl sulfoxide and then diluted in assay buffer as per the inhibitor dilution scheme for Inhibition of human collagenase (MMP-1): Twenty-five microliters of each concentration is added in triplicate to the microfluor plate. The final concentrations in the assay are 30 xcexcM, 3 xcexcM, 0.3 xcexcM, and 0.03 xcexcM.
Substrate (Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2) is prepared as for inhibition of human collagenase (MMP-1) and 50 xcexcl is added to each well to give a final assay concentration of 10 xcexcM. Fluorescence readings (360 nM excitation; 450 emission) are taken at time 0 and every 5 minutes for 1 hour.
Positive controls consist of enzyme and substrate with no inhibitor and blanks consist of substrate only.
IC50""s are determined as per inhibition of human coliagenase (MMP-1). If IC50""s are reported to be less than 0.03 xcexcM, inhibitors are then assayed at final concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.003 xcexcM and 0.0003 xcexcM.
The ability of the compounds or the pharmacoutcally acceptable salts thereof to inhibit the production of TNF and, consequently, demonstrate their effectiveness for treating diseases involving the production of TNF is shown by the following in vitro assay:
Human mononuclear cells were isolated from anti-coagulated human blood using a one-step Ficoll-hypaque separation technique. (2) The mononuclear cells were washed three times in Hanks balanced salt solution (HBSS) with divalent cations and resuspended to a density of 2xc3x97106/ml in HBSS containing 1% BSA. Differential counts determined using the Abbott Cell Dyn 3500 analyzer indicated that monocytes ranged from 17 to 24% of the total cells in these preparations.
180xcexc of the cell suspension was aliquoted into flate bottom 96 well plates (Costar). Additions of compounds and LPS (100 ng/ml final concentration) gave a final volume of 200 xcexcl. All conditions were performed in triplicate. After a four hour incubation at 37xc2x0 C. in an humidified CO2 incubator, plates were removed and centrifuged (10 minutes at approximately 250xc3x97g) and the supernatants removed and assayed for TNFxcex1 using the RandD ELISA let.
For administration to humans for the inhibition of matrix metalioproteinases or the production of tumor necrosis factor (TNF), a variety of conventional routes may be used including orally, parenterally and topically. In general, the active compound will be administered orally or parenterally at dosages between about 0.1 and 25 mg/kg body weight of the subject to be treated per day, preferably from about 0.3 to 5 mg/kg. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The compounds of the present invention can be administered in a wide variety of different dosage forms, in general, the therapeutically effective compounds of this invention are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight.
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with varous disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelation and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For parenteral administration (intramuscular, intraperitoneal, subcutaneous and intravenous use) a sterile injectable solution of the active ingredient is usually prepared. Solutions of a therapeutic compound of the present invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably adjusted and buffered, preferably at a pH of greater than 8, if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Additionally, it is possible to administer the compounds of the present invention topically, e.g., when treating inflammatory conditions of the skin and this may be done by way of creams, jellies, gels, pastes, and ointments, in accordance with standard pharmaceutical practice.
The present invention is illustrated by the following examples, but it is not limited to the details thereof.